Characterization of the electrostatic component of the nucleotide binding to Na,K-ATPase.
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Adenosine Diphosphate↗
Biomedical subjects
M Esmann
Publications and source records attributed to M Esmann.
Structural insights into the binding of cardiac glycosides to the digitalis receptor revealed by solid-state NMR.
Several biologically active derivatives of the cardiotonic steroid ouabain have been made containing NMR isotopes ((13)C, (2)H, and (19)F) in the rhamnose sugar and steroid moieties, and examined at the digitalis receptor site of renal Na(+)/K(+)-ATPase by a combination of solid-state NMR methods. Deuterium NMR spectra of (2)H-labeled inhibitors revealed that the sugar group was only loosely associated with the binding site, whereas the steroid group was more constrained, probably because of hydrogen bonding to residues around the K(+)-channel region. Crosspolarization magic-angle spinning NMR showed that chemical shifts of inhibitors (13)C-labeled in the sugar group moved downfield by 0.5 ppm after binding to the digitalis site, suggesting that the sugar was close to aromatic side groups. A (19)F, (13)C- rotational-echo double-resonance NMR strategy was used to determine the structure of an inhibitor in the digitalis receptor site, and it showed that the ouabain derivatives adopt a conformation in which the sugar extends out of the plane of the steroid ring system. The combined structural and dynamic information favors a model for inhibition in which the ouabain analogues lie across the surface of the Na(+)/K(+)-ATPase alpha-subunit with the sugar group facing away from the surface of the membrane but free to move into contact with one or more aromatic residues.
Carbohydrate Conformation↗
Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation.
Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.
Animals↗
Microsecond motions of the lipids associated with trypsinized Na,K-ATPase membranes. Progressive saturation spin-label electron spin resonance studies.
The microsecond motions of spin-labeled lipids associated with the Na(+)/K(+)-transporting ATP hydrolase (Na,K-ATPase) in native and tryptically shaved membranes from Squalus acanthias have been studied by progressive saturation electron spin resonance (ESR). This includes both the segmental mobility of the lipid chains and the exchange dynamics of the lipids interacting directly with the protein. The lipids at the protein interface display a temperature-dependent chain mobility on the submicrosecond time scale. Exchange of these lipids with those in the bulk bilayer regions of the membrane takes place on the time scale of the nitroxide spin-lattice relaxation, i.e., in the microsecond regime. The off-rates for exchange directly reflect the specificity of ionized fatty acids relative to protonated fatty acids for interaction with the Na,K-ATPase. These essential features of the lipid dynamics at the intramembranous protein surface, namely, a temperature-dependent exchange on the microsecond time scale that reflects the lipid selectivity, are preserved on removing the extramembranous parts of the Na,K-ATPase by extensive trypsinization.
Animals↗
Selectivity of lipid-protein interactions with trypsinized Na, K-ATPase studied by spin-label EPR.
The selectivity of the lipid-protein interactions in trypsinised Na, K-ATPase membranes from Squalus acanthias has been determined by using EPR spectroscopy with different lipid probes spin-labelled on the 14-C atom of the fatty acid chain. From measurements at low ionic strength and different pH values, the pattern of selectivity is: (stearic acid)->(phosphatidylserine)->(stearic acid)0>(phosphatidylcholine)+/-, where superscripts indicate the formal electrostatic charge on the lipid headgroup. This is in the same order as that determined with native Na,K-ATPase membranes [M. Esmann, D. Marsh, Biochemistry 24 (1985) 3572-3578]. The selectivity for phosphatidylserine is independent of pH, over the range pH 6.0-9. 0, as found also for native membranes. For membranes trypsinised in the presence of Rb+ ions, and in the presence of Na+ (which allows more extensive proteolysis), the relative association constants, Kr, of all lipids are the same as for control membranes, with the exception of ionised (stearic acid)- that shows the highest specificity. Therefore, both the stoichiometry and the principal determinants of the specificity of lipid-protein interaction are preserved on extensive trypsinisation of Na,K-ATPase membranes. This has implications for the location and arrangement of those amino acid side chains that determine the lipid selectivity of the native Na,K-ATPase.
Animals↗
Eosin as a probe for conformational transitions and nucleotide binding in Na,K-ATPase.
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Adenosine Diphosphate↗
Nucleotide binding to Na,K-ATPase. Effect of ionic strength and charge.
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Adenosine Diphosphate↗
Characterization of the secondary structure and assembly of the transmembrane domains of trypsinized Na,K-ATPase by Fourier transform infrared spectroscopy.
Fourier transform infrared spectroscopy has been used to compare native Na,K-ATPase-containing membranes with those trypsinized in the presence of either Rb+ or Na+ ions to remove the extramembranous parts of the protein. The protein secondary structure content deduced from the amide I band is approximately 30-35% alpha-helix, 37-40% beta-structure, and 13-15% random coil for native membranes from shark rectal gland and from pig kidney, in both the Na- and K-forms. Trypsinization in either Rb+ (a K+ congener) or Na+ removes approximately 35% of the amide I band intensity of native membranes from shark rectal gland. The protein secondary structural content of the trypsinized membranes lies in the range of approximately 23-32% alpha-helix, 37-46% beta-structure, and 12-18% random coil for the shark and kidney enzymes. The distribution of intensity between the bands corresponding to protonated and deuterium-exchanged alpha-helices, and between the component bands attributed to beta-structure, changes considerably on trypsinization, in the direction of a greater proportion of protonated alpha-helix and a broader range of frequencies for beta-structure. The kinetics of deuteration of the slowly exchanging population of protein amide groups is also changed on trypsinization. The mean rate constant for deuteration of trypsinized membranes is approximately half that for native membranes, whereas the proportion of amides contributing to this population increases on trypsinization. The temperature dependence of the amide I band in the Fourier transform infrared spectra indicates that the onset of thermal denaturation occurs at 58 degrees C for native membranes (in either Na+ or K+) and for membranes trypsinized in Rb+, but the major denaturation event for membranes trypsinized in Na+ occurs at approximately 84 degrees C. These results correlate with the functional properties of the intramembranous section of the enzyme.
Animals↗
The effect of ionic strength and specific anions on substrate binding and hydrolytic activities of Na,K-ATPase.
The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP3-, eosine2-, p-nitrophenylphosphate, and V(max) for Na,K-ATPase and K+-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0-0.5 M of NaCl, Na2SO4, and NaNO3 and in 0.1 M Na-acetate, NaSCN, and NaClO4. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl approximately Na2SO4 approximately Na-acetate < NaNO3 < NaSCN < NaCl04. We treated the influence of NaCl and Na2SO4 on K(diss) for E x ADP as a "pure" ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO3- effect was compatible with competitive binding of NO3- and ADP in addition to the general I-effect. K(diss) for E x NO3 was approximately 32 mM. Analysis of V(max)/K(m) for Na,K-ATPase and K+-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.
4-Nitrophenylphosphatase↗
Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.
Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.
Amino Acid Sequence↗
Influence of Na+ on conformational states in membrane-bound renal Na,K-ATPase.
Conformational states of the Na,K-ATPase and rates of transition between these are studied using the fluorescent dye eosin as a marker for the Na+ form (E1) and occlusion of 86Rb+ as a marker for the K+ form (E2). The aim of the present paper is to propose that the E1 form of the Na,K-ATPase can be liganded with a number of Rb+ and Na+ ions and that only some of these E1 forms bind eosin and thus probably nucleotides with high affinity. Experiments are performed with Na,K-ATPase isolated from pig kidney. Binding of eosin occurs only when Na+ is present at millimolar concentrations, and the observed rate of binding is slow when Rb+ is present. The rate of eosin binding after a sudden increase in the Na+ concentration is about the same as the rate of deocclusion of Rb+, suggesting that eosin monitors the rate of the E2 to E1 transition. Titrations of eosin fluorescence with Na+ indicate that binding of more than one Na+ occurs when high-affinity eosin binding takes place. With 0.05 mM RbCl and 4 mM NaCl present, the Na,K-ATPase is a mixture of at least two enzyme species which do not bind eosin with high affinity. One species is the E2 form with Rb+ occluded, and transition of this form to E1 gives rise to a small observed rate constant for eosin binding when the Na+ concentration is suddenly increased to about 25 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Structural integrity of the membrane domains in extensively trypsinized Na,K-ATPase from shark rectal glands.
Removal of extramembranous portions of the integral membrane protein Na,K-ATPase from shark salt glands by trypsin in the presence of Rb+ (a K+ congener) preserves the intramembranous association of the remaining membrane-spanning tryptic peptides. This is evidenced from comparison of the rotational mobility of native and trypsinized Na,K-ATPase using saturation transfer electron spin resonance spectroscopy (ESR) and from study of the lipid-protein interactions using conventional ESR spectroscopy. The interface between the lipids and the intramembranous domains is conserved on removal of the extramembranous parts of the protein, since the population of motionally restricted boundary lipids remains essentially the same in the native and trypsinized preparations. The ability to occlude Rb+ is also retained by the trypsinized membranes, as previously observed with pig kidney Na,K-ATPase. A 19-kDa fragment remaining when Na,K-ATPase is trypsinized in the presence of Rb+ is degraded further when the trypsinization is carried out in the presence of Na+ instead of Rb+. The rotational mobility of the tryptic fragments in the Na(+)-trypsinized membranes is lower than for the Rb(+)-trypsinized membranes, indicating rearrangement of the peptides. In addition, occlusion capacity is lost when trypsinization is carried out in Na+, suggesting a correlation between structure and function in the trypsinized membranes. The sequences of four membrane-spanning tryptic fragments of shark Na,K-ATPase are found to be almost identical to corresponding sequences in pig kidney Na,K-ATPase.
Amino Acid Sequence↗
Influence of poly(ethylene glycol) and aqueous viscosity on the rotational diffusion of membranous Na,K-ATPase.
The Na,K-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), E.C. 3.6.1.37] in native membranes from the salt gland of Squalus acanthias has been spin-labeled covalently with a chloromercuri nitroxide derivative, and the rotational diffusion of the protein has been studied, as a function of the concentration of glycerol or poly(ethylene glycol) in the suspending medium, by means of saturation-transfer electron spin resonance spectroscopy. The effective rotational correlation time of the protein increases linearly with the viscosity of the aqueous glycerol medium, with a gradient whose value indicates that ca. 50-70% of the volume of the Na,K-ATPase protein is external to the membrane. The effective rotational correlation times of the protein in poly(ethylene glycol) solutions are considerably greater than those in glycerol solutions of the same viscosity and increase nonlinearly with the viscosity of the suspending medium, indicating that increasing concentrations of poly(ethylene glycol) induce aggregation of the integral proteins within the membrane. The value reached at 50% poly(ethylene glycol) corresponds to a degree of aggregation of the proteins between 2 and 5 depending on whether the ethylene glycol polymer is excluded from the membrane surface region. The results are discussed with respect to hydration forces and poly(ethylene glycol)-induced cell fusion.
Animals↗
Ultrastructure of membrane-bound Na, K-ATPase after extensive tryptic digestion.
Membrane-bound Na, K-ATPase was digested with trypsin in the presence of Rb+ to form the stable 19-kDa and smaller fragments of the alpha-chain known to preserve occlusion of Rb+ (K+) or Na+. The trypsinized membranes obtained from pig kidney and shark rectal gland were analyzed by electron microscopy. Tryptic digestion preserved general membrane structure but removed both the surface particles observed by negative staining and the protruding cytoplasmic portion of the alpha-subunit identified in thin sections. However, intramembrane particles defined by freeze-fracture were preserved after trypsinization suggesting that the remaining membrane spanning protein fragments retain the native structure within the lipid bilayer after proteolysis.
Animals↗
Maleimide, iodoacetamide, indanedione, and chloromercuric spin label reagents with derivatized nitroxide rings as ESR reporter groups for protein conformation and dynamics.
The syntheses of eight nitroxide spin labels which bear maleimide, iodoacetamide, indanedione, or chloromercuric reactive groups and, in addition, a second substituent in the nitroxide ring are presented. The second substituent groups range from hydrophobic and hydrophilic esters to carboxylic acid and secondary and tertiary amine groups. The resulting spin labels are characterized with respect both to protein covalent modification and to the electron spin resonance spectral properties of the bound labels. The effect of the various substituents in the spin label on the reactivity toward the membrane-bound shark rectal gland and pig kidney Na,K-ATPase is described. The spectral differences between immobilized and mobile groups observed by electron spin resonance for the different protein-bound spin labels show that, by selecting an appropriate derivative for modification, a large range of different motional sensitivities of the reporter group can be obtained. Such different series of spin labels should therefore be useful for detecting mobility changes arising from conformational transitions in proteins by conventional electron spin resonance spectroscopy or for measurement of protein rotational diffusion using saturation transfer electron spin resonance spectroscopy. The chloromercuric series is found to be particularly useful because of the high reactivity, the lack of reversibility that potentially is associated with the Michael addition reaction, and the wide range of rotational mobility that is exhibited by the different derivatives.
Animals↗
Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives.
Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.
Animals↗
Determination of rate constants for nucleotide dissociation from Na,K-ATPase.
A method for determining individual rate constants for nucleotide binding to and dissociation from membrane bound pig kidney Na,K-ATPase is presented. The method involves determination of the rate of relaxation when Na,K-ATPase in the presence of eosin is mixed with ADP or ATP in a stopped-flow fluorescence apparatus. It is shown that the nucleotide dependence of this rate of relaxation--taken together with measured equilibrium binding values for eosin and ADP--makes possible a reasonably reliable determination of the rate constant for dissociation of nucleotide, i.e., determination of the rate constant k-1 in the following model (where E denotes Na,K-ATPase): [formula: see text] All experiments are carried out at about 4 degrees C in a buffer containing 200 mM sucrose, 10 mM EDTA, 25 mM Tris and 73 mM NaCl (pH 7.4). Values obtained for the rate constants for dissociation are about 6 s-1 for ADP and 2-3 s-1 for ATP.
Adenosine Diphosphate↗
Conventional and saturation transfer EPR spectroscopy of Na+/K(+)-ATPase modified with different maleimide-nitroxide derivatives.
The membranous Na+/K(+)-ATPase from Squalus acanthias has been covalently modified on either Class I or Class II sulphydryl groups using derivatives of 3-(maleimidomethyl)-1-oxyl-2,2,5,5-tetramethylpyrrolidine with substituents of different charge and hydrophobicity attached at the remaining unsubstituted position of the pyrrolidine ring. The substituent groups used were a methyl and a hexyl ester, and di- and tri-methylammonium ethyl esters, as well as the parent underivatized compound. Additionally, another series of maleimide-nitroxides differing (by zero to seven intervening atoms) in the length of the linking group between the maleimide and the pyrrolidine moieties was used. The sites of attachment have been characterized in terms of the rotational mobility and environmental polarity by using conventional and saturation transfer EPR spectroscopy of these spin-labelled reagents. This provides a further sub-classification of the primary Class I and Class II SH-groups on the alpha-subunit of the enzyme, which differ both in their reactivity and influence on the Na+/K(+)-ATPase activity.
Animals↗